Apparatus And Methods For Concentrating And Separating Particles Such As Molecules

ABSTRACT

Particles of interest, such as DNA molecules, are injected into a medium by applying a first field. Once in the medium the particles are concentrated by applying one or more fields that cause mobilities of the particles in the medium to vary in a manner that is correlated with motions of the particles. Particle injection and particle concentration may be performed concurrently or in alternation.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority from Canadian patent application No.2,496,294 filed on 7 Feb. 2005. The subject matter of this applicationis related to the subject matter of U.S. patent application No.60/540,352 filed on 2 Feb. 2004 and PCT patent application No.PCT/CA2005/000124 entitled “Scodaphoresis and Methods and Apparatus forMoving and Concentrating Particles” filed on 2 Feb. 2005, both of whichare hereby incorporated herein by reference.

FIELD OF THE INVENTION

The invention provides methods and apparatus for guiding the motions ofparticles, such as molecules, for example, DNA, RNA, proteins and otherbiomolecules. The invention may be applied in systems for concentratingmolecules and/or separating molecules of different types, lengths and/orphysical or chemical characteristics. Some applications involveselectively trapping particles in a gel material that is subjected tocontinuous or pulsed electrokinetic injection of a sample.

BACKGROUND

There are many fields in which it is desirable to concentrate particlesso that the particles can be studied. Consider for example the widerange of fields in which it may be desirable to collect molecules of DNAfor study. Such fields include crime detection, medical studies,paleology, environmental studies and the like. The DNA of interest maybe present initially in exceedingly low concentrations. There is a needfor practical ways to concentrate particles, such as DNA.

SUMMARY OF THE INVENTION

This invention has a number of aspects. One aspect of the inventionprovides a method for concentrating selected particles. The particlesmay, for example, comprise DNA molecules, RNA molecules or denaturedproteins. In specific embodiments of the invention the particlescomprise DNA. The method comprises providing particles, including theselected particles, in a first region and applying a first fielddirected to move at least the selected particles from the first regioninto a second region. At least the second region is a region of a mediumin which a mobility of the selected particles is dependent on anintensity of one or more second fields. When the selected particles arein the second region the method proceeds by concentrating the selectedparticles in a vicinity of a point in the second region by applying thesecond fields.

Another aspect of the invention provides a method for concentratingparticles of interest. The method comprises driving the particles into amedium by applying a particle-injecting electric field across a boundarybetween the medium and a sample containing the particles of interest;and, applying scodaphoresis to the particles of interest in the mediumto concentrate the particles of interest at a location in the medium.

Another aspect of the invention provides apparatus for concentratingparticles of interest. The apparatus comprises: a buffer reservoircapable of receiving a sample; a medium in which particles of interesthave a mobility that depends upon the intensity of applied fields; meansfor applying a first field to drive particles of interest from thebuffer reservoir into the medium; and, means for applying one or moresecond fields to concentrate the particles of interest at a focal spotwithin the medium.

Further aspects of the invention and features of embodiments of theinvention are described below.

DESCRIPTION OF THE FIGURES

The attached Figures are intended to aid in the visualization ofpotential embodiments of the technology, they should not be interpretedas limiting with respect to the scope of the invention described herein.

FIG. 1 is a schematic view of a SCODA setup having 4 large bufferreservoirs around 4 sides of a gel;

FIG. 2 is a schematic view of a gel cast with buffer reservoirs on twosides.

FIG. 3 shows apparatus for performing SCODA and electrokinetic injectionfrom a sample reservoir.

FIGS. 4A through 4D illustrate the operation of a method according tothe invention.

FIGS. 5A and 5B illustrate the deflection of SCODA focused DNA spots byapplication of a DC field.

FIGS. 6A, 6B and 6C illustrate separation of particles that have beenconcentrated at a focal spot by a one-dimensional separation technique.

DESCRIPTION

SCODAphoresis (hereinafter referred to as SCODA) is described in U.S.patent application No. 60/540,352 filed 2 Feb. 2004, PCT patentapplication No. PCT/CA2005/000124 entitled “Scodaphoresis and Methodsand Apparatus for Moving and Concentrating Particles” filed on Feb. 2,2005; and Marziali, A.; et al., “Novel electrophoresis mechanism basedon synchronous alternating drag perturbation”, Electrophoresis 2005, 26,82-89 all of which are hereby are incorporated herein by reference.Since SCODA is described in these published materials it is notdescribed in detail herein.

SCODA is a process that can be used for concentrating particles (whichmay consist of or include certain molecules, such as DNA). SCODA can beused to concentrate the particles in the vicinity of a point in a regionof a suitable material in which the particles have mobilities that varyin response to an applied field or combination of applied fields. Wherethe particles are electrically-charged molecules, such as DNA, theapplied fields may comprise electric fields. The material may comprise asuitable gel such as an agarose gel, for example.

SCODA does not require electrodes to be present at the location whereparticles are concentrated. In one embodiment SCODA provides focusingand concentration of molecules based on the non-linear dependence of theparticles' velocity on the strength of an applied electric field. Thiscan also be stated as being based on the field dependence of theparticles' mobility. The velocity, v of a particle in an electric fieldcan be expressed as:

{right arrow over (v)}(t)=μ(E)·{right arrow over (E)}  (1)

where μ is the mobility of the particle and E is the magnitude of theapplied electric field. In some media the mobility μ is reasonablyapproximated by:

μ(E)=μ₀ +kE  (2)

where μ₀ and k are constants. In such media, the particle velocityvaries non-linearly with the magnitude of the applied electric field.

Under the application of SCODA fields, molecules for which the value ofk is large have a greater tendency to focus than particles with smallervalues of k. In one embodiment of SCODA, a sample containing particlesof interest mixed with other particles is introduced into a gel. Thematerial of the gel and/or SCODA fields are selected so that theparticles of interest have large values for k while other particlespresent in the gel have smaller values for k. When SCODA fields areapplied, the particles of interest tend to be focused in a spot at alocation determined by the SCODA fields. Molecules with low values for kremain distributed throughout the gel.

This effect is also impacted by the ability of the molecules to diffusein the gel. The SCODA velocity toward the center of the gel isproportional to k and to the distance r from the location at which themolecules become concentrated. In an ideal case where the molecules ofinterest have mobilities given by Equation (2) it can be shown that:

$\begin{matrix}{{\overset{->}{v}} = {{- \frac{{kEE}_{q}}{4}}r}} & (3)\end{matrix}$

where v is the average velocity of the molecules in a direction of thefocal point around which the molecules become concentrated, E is themagnitude of the SCODA electric field, and E_(q) is the charge on themolecules.

The ability of molecules to focus (e.g. 1/radius of the focused spot) isproportional to:

$\begin{matrix}\sqrt{\frac{k}{D}} & (4)\end{matrix}$

where D is the diffusion constant of the molecules in the gel (or othermedium). Particles with a large value of this parameter tend to focus inthe vicinity of a point in the gel under SCODA conditions, and areselectively concentrated relative to concentrations of other moleculeswith a smaller value of this parameter.

Particles may be injected into a region of a medium within which theparticles can be concentrated by SCODA by providing the particles in anadjacent region and applying a field that causes the particles to moveinto the region of the SCODA medium. The adjacent region may be called afirst region and the region of the SCODA medium may be called a secondregion. The field that causes the particles to move from the firstregion into the second region may be called a first field. The firstfield may comprise any field to which particles of interest respond bymoving. Where the particles are electrically charged, the first fieldmay comprise an electric field.

Depending upon the nature of the particles of interest, the first fieldmay comprise any of:

-   -   a magnetic field;    -   an electric field;    -   a flow field; or,    -   some combination thereof.

In one embodiment, DC (Direct Current) electrophoresis is used tointroduce particles from a sample into a SCODA medium such as a precastgel. After particles have been introduced into the gel, SCODA can beapplied to concentrate selected particles at a location in the gel. DCelectrophoresis may be applied to drive particles from a flowing sampleinto a SCODA medium.

The sample may comprise a liquid in which the particles are entrained.In some embodiments a liquid sample is introduced into a chamberadjacent to the SCODA medium and particles are driven from the chamberinto the SCODA medium by electrophoresis until a desired quantity ofparticles are present in the SCODA medium or until the sample isdepleted of particles. In other embodiments the sample is changed eithercontinuously or intermittently and the first field is applied eithercontinuously or intermittently to inject particles from the sample intothe SCODA medium. Changing the sample may comprise intermiftentlyremoving some or all of the sample and replacing the removed sample withfresh sample. In some embodiments, changing the sample comprisesallowing a liquid, which constitutes the sample, to flow through achamber adjacent to the SCODA medium.

In cases where the sample is replenished, particles of interest thatoccur in the sample in exceedingly small concentrations can be collectedat the focus in the SCODA medium over time. A very large concentrationfactor can be achieved in this manner.

Where the first field comprises a DC electrophoresis field, the fieldmay be such that only certain charged species of interest are extractedfrom sample and introduced into the medium. For example, in someembodiments, DC electrophoresis is used to carry charged molecules whichinclude nucleotide polymers, such as DNA, into the gel or other SCODAmedium. Particles that are not charged or particles that have charges ofthe opposite polarity to the desired charged molecules are not movedinto the SCODA medium.

It is not necessary that the magnitude of the first field be constant oreven that the first field always have the same polarity. All that isrequired is that there is a net flow of particles of interest into theSCODA medium under the influence of the first field.

SCODA is performed by applying one or more SCODA fields within the SCODAmedium. As described in PCT patent application No. PCT/CA2005/000124,for appropriate selection of SCODA fields, particles and SCODA media,the application of the SCODA fields causes selected particles within theSCODA medium to converge to a focal point so that the selected particlesbecome concentrated in a vicinity of the focal point. The SCODA fieldsmay be called “second fields”. The second fields may co-exist with thefirst field in any of a number of ways including:

-   -   The first field is superposed on the second field(s) such that        the first and second fields are applied simultaneously; or,    -   The first field is interspersed in time with the second field(s)        such that only the first field is applied for a first period of        time, and only the second field(s) is applied for a second        period of time. This pattern may be repeated at least until the        selected molecules or other particles from the sample have been        introduced into the SCODA medium; or,    -   Some combination of these, for example, the first field may be        applied during selected portions of a cycle of the second        field(s) or the first field may be applied both during selected        portions of a cycle of the second field (s) and also during        periods when the second field(s) is not being applied.

The use of a DC field to inject particles into a SCODA medium permits:

-   -   Gel for use as a SCODA medium may be cast in relatively pure        buffer, rather than in sample (which may be sufficiently        contaminated to preclude satisfactory gel casting);    -   Only molecules of one charge species enter the gel in the first        place, so neutral, and oppositely charged molecules are left        behind and do not contaminate the gel;    -   Only the desired molecules with high values of k/μ₀ are trapped.        These can be extracted by shutting off the DC field, allowing        the focus to move to the center, and performing any suitable        extraction method.    -   The effective value for k for some molecules such as DNA can be        made different for different sizes (e.g. lengths) of molecule by        adjusting the frequency of the SCODA driving field(s).

Particles that have become concentrated in the vicinity of a point inthe SCODA medium may be extracted in any suitable manner including:removing a portion of the gel or other SCODA medium that contains theconcentrated particles; or causing the concentrated particles to moveout of a plane of the SCODA medium by applying electric or other fields;or the like.

Any suitable combination of fields may be used to provide SCODA focusingof molecules. It is not necessary that the SCODA use electric fields.This invention can be applied to any system that employs SCODA in any ofthe embodiments described in the above referenced SCODA patentapplications.

Example Operation of the Invention

FIG. 1 shows example apparatus 10 for performing concentration by SCODA.Apparatus 10 includes a sheet 14 of gel medium located amid bufferreservoirs 12A to 12D (collectively buffer reservoirs 12). One bufferreservoir is on each side of gel 14. Electrodes 13A to 13D are eachimmersed in a corresponding one of the buffer reservoirs. Electrodes 13Ato 13D (collectively electrodes 13) are connected to different channelsof a programmable power supply that applies potentials to electrodes 13to provide a SCODA field in gel 14. Under the influence of SCODA fields,mobile particles in gel 14 (such as molecules) remain nearly stationaryif they have values of key≅0. All appropriately-charged molecules withnon-zero k move toward a central focus 16 as indicated by arrows 17. Forexample, SCODA fields may be provided that cause negatively-chargedparticles to move toward central focus 16 while positively-chargedparticles move away from central focus 16, or vice versa.

Particles may be introduced into gel 14 by introducing the particlesinto the buffer in one of buffer reservoirs 12 (for example, bufferreservoir 12A) and applying a potential difference between thecorresponding electrode 13 and one or more other ones of electrodes 13to create a first electric field directed to cause particles, which maybe molecules in the buffer reservoir 12, to move toward gel 14.Typically the first electric field is created by establishing apotential difference between two electrodes that are on opposite sidesof SCODA medium 14 (for example, between electrodes 13A and 13C). ThisDC field has a polarity selected so that charged molecules or otherparticles of interest will be injected from the buffer 12 into the SCODAgel 14. The DC field may be an applied electric field of the typecommonly used in DC electrophoresis.

In buffer 12, the particles move toward gel 14 with a velocityproportional to their mobility in the buffer, μ_(BUF), until they entergel 14. Within gel 14, the particles follow a combined motion withmobility μ₀ (which will typically be different from μ_(BUF)) withrespect to the first field, and, while the second (SCODA) field(s) isapplied, with an effective mobility proportional to k with respect tothe second field(s).

Once in gel 14 (or other medium), particles having low values for k willbehave as in DC electrophoresis, and will migrate through gel 14. If thefirst field is applied for long enough, such molecules may traversecompletely across gel 14 until they escape into the buffer reservoir 12opposed to the buffer reservoir 12 from which they originated.

Particles with high values of k will be focused by the SCODA field oncethey have entered gel 14 and will be trapped in gel 14 (as long as thefirst field—which may be a DC electric field—is not so strong as tooverwhelm the SCODA velocity given to such particles). The location ofthe focus at which particles become concentrated will be shifted fromthe location of the focus in the absence of the first field. The amountof shift is based on the ratio of μ₀/k and on the relative amplitudes ofthe first and second fields. For some particles k may befrequency-dependent. In such cases the amount of shift may also dependupon the frequency of the second field(s).

The buffer reservoir 12 into which particles are introduced need not belarge and could be a buffer-filled space between an electrode and mediumin a typical SCODA apparatus like apparatus 10 of FIG. 1. If theparticles are of a type that could be damaged by electrochemicalreactions at the electrode then the particles should be introduced at alocation such that the particles do not need to pass by the electrodebefore entering the medium. For example, the particles could beintroduced into a region between the electrode and the medium. Providinga larger buffer reservoir and/or a buffer reservoir that permits fluidto be circulated permits extracting particles of interest from largersample volumes and makes possible greater degrees of concentration.

FIG. 2 is a schematic view of apparatus 20 comprising a region of a gel14 cast with buffer reservoirs 12A and 12B on two opposed sides.Apparatus 20 is similar to a conventional electrophoresis apparatus. ADC field is created by applying a potential difference betweenelectrodes 13A and 13B. A sample containing molecules or other particlesof interest is placed in one of the buffer reservoirs 12. Appropriatepolarity of the DC field causes molecules of a desired charge to entergel 14 from the sample. The molecules typically have mobilities μ_(BUF)in the buffer reservoir 12 that are significantly greater than theirmobilities μ₀ in gel 14. This causes molecules to initially stack at theedge of gel 14 and then separate into bands of different mobility.Typically, differently sized molecules travel in bands through the gelat different velocities. By applying SCODA fields when particles ofinterest are in gel 14, the particles of interest can be made to collectin the vicinity 16 of a focal point. Other particles pass through gel 14into the opposing buffer reservoir 12. A mechanism 22 applies suitableSCODA fields within gel 14.

FIG. 3 shows apparatus 30 which combines features for efficientlyperforming electrophoretic injection of molecules from a sample into amedium and subjecting molecules in the medium to SCODA. Apparatus 30 issimilar to apparatus 10 FIG. 1 except that one buffer reservoir 32A ismade substantially longer than the other buffer reservoirs 32B through32D to allow a significant volume of a sample S containing particles Pto be injected. Each buffer reservoir is in electrical contact with acorresponding electrode 33. Electrode 33C is located at an end of bufferreservoir 32A so that buffer reservoir 32A and SCODA medium 34 liebetween electrodes 33A and 33C.

Particles P can be driven into medium 34 by applying a potentialdifference between electrodes 33A and 33C with a power supply 35. Thepotential difference causes particles P to move into medium 34 asindicated by arrow 37.

Power supply 35 is also capable of applying time-varying potentials toelectrodes 33 to cause SCODA fields within medium 34. The SCODA fields,when present, cause selected particles within medium 34 to convergetoward the vicinity 40 of a focal point as indicated by arrows 39.

It is preferable to provide SCODA electric fields using electrodes thatare located symmetrically relative to medium 34. Apparatus 30 has asensing electrode 42 provided at a location that is symmetrical withrespect to electrode 33C. If electrode 42 were used as a SCODA electrodethen the sourcing or sinling of current at electrode 42 could damageparticles P as they pass by electrode 42. Sensing electrode 42 providesa feedback signal 43 to power supply 35. Power supply 35 receives signal43 at a high impedance input so that virtually no current is sourced orsinks at electrode 42.

Power supply 35 controls the potential applied to electrode 33A basedupon feedback signal 43 to cause the potential at electrode 42 to tracka desired SCODA waveform. This may be accomplished by providing acontroller which uses a difference between the potential sensed atsensing electrode 42 and the desired SCODA potential as negativefeedback. The potential at electrode 42 can be controlled at a desiredvalue by appropriately regulating the potential applied to electrode33A. Thus sensing electrode 42 in combination with the control in powersupply 35 serves as a virtual SCODA electrode. This permits attainmentof the proper SCODA field even though electrode 33A is displaced fromits ideal position adjacent to medium 34.

In some cases providing a sensing electrode 42 closer to medium 34 andusing a sensed voltage 43 to control the potential on a current-sourcing(or current-sinking) electrode farther from the medium (such aselectrode 33A) can help to make the SCODA fields independent of theelectrical conductivity of sample S. Electrical conductivity ofdifferent samples may vary due, for example, to variations in salinitybetween the samples.

Apparatus like that of FIG. 3 may be used to concentrate selectedmolecules that are present in sample S by applying particle-injectingand SCODA fields in alternation. For example, a DC field may be appliedbetween electrodes 33A and 33C such that particles P such as chargedmolecules of interest move from sample reservoir 32A into medium 34(this phase may be termed DC injection). DC injection is performed atleast until molecules of interest in the buffer are significantlyremoved from the area around electrode 33A.

At the end of sample buffer 32A that adjoins medium 34 molecules areinjected into medium 34. After an appropriate time, the DC field is shutoff, and the SCODA field is turned on. Preferably the SCODA field is notturned on until after the molecules of interest have been driven farenough from electrode 33A that any reverse DC field temporarily appliedduring SCODA operation does not drive molecules into electrode 33A wherethey may be chemically altered. To ensure this, the amount of time thatthe DC field and SCODA fields are applied for should obey:

T_(INJ)μ_(BUF)E_(DC)>t_(SCODA)μ_(BUF)E_(SCODA)  (5)

where: T_(INJ) is the length of time that the DC injection field isapplied; E_(DC) is the magnitude of the DC injection field; t_(SCODA) isthe length of the time interval in one SCODA cycle during which areverse field is applied between electrodes 33A and 33C, and E_(SCODA)is the magnitude of the SCODA field applied in that interval. In anexample embodiment of SCODA, t_(SCODA) is three seconds, or ¼ of theduration of the SCODA cycle.

In embodiments wherein SCODA and the DC injection fields are interleavedin time, once the SCODA field has been applied for an appropriate amountof time it is shut off and the DC injection can be resumed to introducefurther charged molecules into the gel. The cycle can be repeated toenhance the concentration of selected molecules at focus 40.

It is also possible to apply the DC injection field in superpositionwith the SCODA field. In this case, particles P will enter medium 34 andbe focused by SCODA at the same time. Focus spot 40 will continue toincrease in molecule concentration, as long as more molecules P areavailable in buffer reservoir 32A, and as long as the DC injection fieldis at least periodically or sporadically turned on to cause molecules Pto be injected into medium 34. When SCODA and DC fields are appliedsimultaneously, the DC field will cause the location of focus spot 40 tobe pushed away from the location that focal spot 40 would have in theabsence of the DC injection field.

An estimate of the amount that focal spot 40 is shifted by a DC fieldcan be made using analytic approximations to the SCODA velocity ofEquation (3) and the approximation of the drift velocity of theparticles in the DC injection field of:

|{right arrow over (v)}|=μ₀E_(DC)  (6)

Both of these velocities are taken in the horizontal direction. The DCdrift will cause the focus to shift to a location where these velocitiesare equal and opposite. This will occur at:

$\begin{matrix}{r = {4\frac{\mu_{0}E_{DC}}{{kEE}_{q}}}} & (7)\end{matrix}$

in other words, the focus location will be based on the applied fields,and on the ratio of μ₀/k for the molecules. This yields the additionaladvantage that molecules may be separated according to the parameterμ₀/k. Typically, for DNA, μ₀ decreases with increasing length, while kincreases with increasing length. Clearly, longer molecules will tend tofocus nearer to the center of medium 34 with both SCODA and DC injectionfields applied, and shorter molecules will focus closer to the edge ofmedium 34. By increasing the DC field, one can push the foci at whichsmaller molecules collect off the edge of medium 34 to remove suchsmaller molecules from medium 34. This mechanism can be applied forenriching a focal spot 40 with large DNA and also for separating DNA orother molecules with small μ₀/k from molecules with large μ₀/k (ions,possibly proteins fall in this latter category).

In embodiments wherein the SCODA and DC injection fields occur at thesame time, it is desirable to start DC injection first for a periodsufficient that the condition of Equation (5) is satisfied. Themolecules of interest in buffer reservoir 32A will then be sufficientlyfar from electrode 33A that they will not be driven into electrode 33Aby the SCODA field.

An electrically conducting fluid barrier 44, such as a barrier made ofgel, may optionally be placed between electrode 33A and sample S toavoid the possibility that particles in sample S will contact electrode33 by convective mixing or otherwise.

By applying DC electrokinetic injection during SCODA operation, theconcentration factor achievable by SCODA can be increased by injectingfor longer, rather than or in addition to using a bigger medium 34.

Apparatus 30 comprises a source 48 of sample S that can be introducedinto buffer reservoir 32A by way of valve 50 and pump 52. Excess sampleS can escape from buffer reservoir 32A by way of overflow 54. Additionalparticles P can be made available for concentration in focal spot 40 byperiodically or continuously operating pump 52 to introduce fresh sampleS into buffer reservoir 32A.

Apparatus 30 also includes a source 58 of clean buffer solution B. Whena desired amount of particles P have collected at focal spot 40, sampleS can be purged from buffer reservoir 32A by switching valve 50 andoperating pump 52 to flush buffer reservoir 32A with buffer B. Continuedapplication of the DC injection field after sample S has been removedfrom buffer reservoir 32A causes those particles P that are not trappedat focal spot 40 by application of the SCODA fields to be washed out ofmedium 34 into buffer reservoir 32C.

Where SCODA can achieve a spot radius of 200 μm, performing SCODA on a 1cm by 1 cm gel medium 34 in which particles P are initially evenlydistributed (as would be the case, for example, if the sample is cast aspart of the gel) provides a concentration factor of 800. Adding a 10 cmlong sample reservoir next to the SCODA gel and performing DC injectionwhile SCODA is running can increase the concentration factor to 8,000.Once the sample reservoir is depleted of particles of interest, it canbe drained and replenished with more sample and run again. Each run addsto the concentration factor. Concentration factors in excess of 10,000times have been achieved.

The methods described herein can be used to collect particles ofinterest from extremely dilute samples. For example, in one experiment,a sample was made up by diluting approximately 10 molecules of DNAhaving a target sequence into 5 ml of buffer. The resulting solution (inwhich the DNA had a zeptomolar −10⁻²¹ M concentration) was subjected toDC injection and SCODA as described herein. A plug of gel was removed atthe SCODA focal spot. The plug of gel was subjected to a SYBR greenchemistry RT-PCR reaction. The target DNA sequence was identified in theresulting amplified DNA.

Apparatus according to the invention may comprise appropriate pumps andvalves to repeatedly draw fresh sample into the buffer sample reservoir,inject until depleted or substantially depleted of molecules (or otherparticles) of interest, then drain and renew with fresh sample. Suchpumps and valves may be operated automatically under control of anautomatic controller such as a computer, PLC, hard-wired logic circuit,some combination thereof, or the like.

In a prototype demonstration of DC electrokinetic injection, a DCinjection field was applied until the buffer reservoir was depletedbefore running the SCODA field. FIGS. 4A to 4D schematically demonstratethis process. Electrodes are not shown in FIGS. 4A to 4D. In FIG. 4A, asample S is in a buffer reservoir 32A adjoining a SCODA medium (such asa gel) 34. Sample S contains particles of interest.

In FIG. 4B, a DC injection field 60 is applied. The DC injection fieldcauses particles to move from reservoir 32A into medium 34. In FIG. 4C,SCODA fields have started to concentrate the particles at a focal spot.In FIG. 4D, continued application of the SCODA fields has caused theparticles to be concentrated at a focal spot 62.

In a separate experiment, SCODA was run while the DC field was appliedto observe the deflection of the focal spot. FIG. 5A illustrates thelocation of focal spot 62 when SCODA fields are applied in the absenceof an injection field. FIG. 5B shows how focal spot 62 is displaced by adistance r when a DC injection field 60 is superposed on the SCODAfield.

Sometimes the particles that are concentrated in a focal spot are ofdifferent species. The methods may optionally include a step to separatethe species that have collected at a focal spot. The separation maycomprise a one-dimensional separation. Methods that may be used toseparate the species at a focal spot include-electrophoresis.

In some embodiments, separation is performed by applying a DC field thattends to move particles from the focal spot in one direction andapplying an alternating field having a magnitude that is significantlygreater in one polarity than the other but an average value thatintegrates to approximately zero (a ZIFE field). The alternating fieldis arranged so that it tends to move the particles in the oppositedirection to the DC field.

Under the influence of a DC field, particles move at velocitiesdetermined primarily by the value of μ₀. Under the influence of the ZIFEfield, the particles move at net drift velocities determined primarilyby the value of k.

For different species of particle, one or the other of the two fieldswill dominate. Depending upon which of the fields dominates, theparticles will move away from the focal spot in one direction or theother. Which of the fields dominates for a particular species willdepend upon the value of k/μ₀ for that species. The result of separationis a smear or a series of spots spread out along a line as indicated inFIG. 6C. For a species having a given value of k/μ₀ it is possible tochoose fields so that the effect of the DC and alternating fields on thespecies is balanced. In this case, the species will stay at the focalspot.

In general, for DNA, k increases with molecular weight and μ₀ decreaseswith molecular weight. Therefore, separating species of DNA based uponthe ratio k/μ₀ generally corresponds to separating the DNA species bymolecular weight.

Though some drift of desired bands will likely occur, careful mapping ofmolecular weight to DC/ZIFE field ratios may allow for removal of DNAfragments outside a relatively tight molecular weight range by selectingconditions which will result in molecules of a particular weight stayingat the focal spot and extracting the center of the focal spot after thelinear spreading process has been proceeding for sufficient time to moveother species away from the focal spot. If enrichment of high molecularweight DNA is desired, the fields are chosen such that the species thatis at equilibrium (i.e. does not move away from the focal spot) has alarge value of k/μ₀ so that DNA having the highest molecular weight lagsnear the focal spot. Though this may appear similar to DCelectrophoresis, it should be noted that, since the average velocity ofthe desired band is near zero, substantial separation can be achieved ina short distance, though possibly over a long time.

As shown in FIGS. 6B and 6C a spot 70 of a marker, such as a DNA ladder,may be applied adjacent a focal spot 72. After separation, species inthe focal spot and ladder are separated. During the separation step, theladder separates into spots 74 containing species having known lengthsor other characteristics. These spots can be correlated to spots 76resulting from the separation of species in focal spot 72 to identifycharacteristics of the species in focal spot 72.

In some embodiments, the locations of one or more specific marker spots74 are monitored. The position of a marker spot can be used to controlthe ratio of DC/ZIFE fields, to stabilize the location of a band ofinterest. The controller may control a magnitude of one of the DC andZIFE fields or may control magnitudes of both the DC and ZIFE fields. Inone embodiment, a marker spot 74 contains particles which have the sameproperties as particles being screened for and the controller usesnegative-feedback control to maintain the marker spot 74 stationary. Theposition of a marker spot 74 may be monitored by a machine visionsystem, for example.

As will be apparent to those skilled in the art in light of theforegoing disclosure, many alterations and modifications are possible inthe practice of this invention without departing from the spirit orscope thereof. For example:

-   -   An asymmetrical alternating electric field could be used to        drive particles of interest into a medium if the field is        selected so that the particles of interest are driven farther        into the medium on portions of the cycle which tend to move        particles into the medium than the particles of interest are        moved back toward the sample on portions of the cycle during        which the alternating particle injecting field causes the        particles to move back toward the sample.    -   A membrane or other barrier that is electrically conducting but        blocks the passage of the particles may be disposed between the        electrode in electrical contact with the sample (e.g. electrode        13C) and the medium. The sample may be introduced between the        barrier and the medium. This prevents the particles of interest        in the sample from contacting the electrode.    -   An electrode could be provided within the medium and used for        the purpose of injecting particles of interest into the medium.        The electrode could be disconnected or removed during SCODA (at        least before particles of interest could reach the electrode).        Such an electrode could, for example, be located in a well        located in a central region of the medium. Such an electrode        could provide a radial electric field having a polarity to        inject particles-of-interest into the medium. With this        arrangement, particles could be injected into the medium from        any or all sides of the medium.    -   Sample could be introduced out of the plane of a sheet-like        medium. For example, a layer of fluid containing a sample could        be placed on a layer of gel. Particles (such as molecules of        interest) could be driven from the fluid into the gel by        applying a particle-injecting electric field having a component        normal to the surface of the gel and a polarity appropriate to        cause the molecules to enter the gel. The electrode(s) used to        apply the particle-injecting field could be removed prior to        commencing the application of SCODA fields if the presence of        such electrode(s) would undesirably disrupt the SCODA fields.        The layer of fluid containing the sample could also be removed        after the particles-of-interest have been injected into the        medium, if desired.    -   A fluid sample containing particles of interest could be caused        to flow through a passageway bounded at least in part by the        medium. A particle-injecting field could be applied to cause        particles of interest to enter the medium from the fluid flowing        in the passageway. Such an embodiment could be applied, for        example, to environmental monitoring.    -   A reservoir containing a sample does not need to be external to        the medium but could comprise a passage or other chamber within        the medium. For example, the methods of the invention could be        applied to inject particles-of-interest into a medium from a        passageway extending partially or entirely within the medium        along an edge thereof.

Those skilled in the art will recognize that the technology describedherein has a wide range of applications, including applications such as:

-   -   Extracting DNA from soil—A device could apply the methods        described herein to extract target DNA of interest from soil        samples for applications such as forensics, environmental        pathogen detection, or metagenomics studies. A soil sample could        be resuspended in a buffer/lysis solution and added to a buffer        reservoir of apparatus as described herein. DC loading fields        could be applied across the buffer reservoir to load the DNA        into a separation matrix, then concentration fields applied to        concentrate the DNA to a focal spot for extraction. The        concentrated DNA could then be added to a quantitative real-time        PCR reaction with target-specific probes to detect the absence        or presence and amount of target DNA sequences in the sample.    -   Extracting DNA from large volumes of solution—apparatus having a        flow-though pumping system as shown, for example, in FIG. 3        could be used to continually cycle sample through a buffer        reservoir, therefore permitting concentration of nucleic acids        from an arbitrarily large sample volume. In an alternative        embodiment, the apparatus may lack a separate buffer reservoir        for receiving sample. Injection electrodes could be positioned        in such a way to load particles of interest directly into a        SCODA medium from a large volume of solution. For example,        apparatus could be placed in a water reservoir. An electrode        could be provided to load DNA from the reservoir into a SCODA        medium. Such apparatus could be used to test water reservoirs        for contamination such as E. coli. In some cases, real-time        detection could be built into a single system to integrate the        concentration and detection of target sequences.    -   Concentrating nucleic acids from airborne pathogens—Air        particulate filters, such as those used on airplanes, may be        rinsed in solution to release collected airborne pathogens        and/or viruses into the solution. The nucleic acids can then be        lysed and concentrated. A target-specific detection technique        can then be used to rapidly identify targets such as SARS, avian        flu, anthrax, or the like.    -   RNA concentration to determine gene expression—Cellular lysate        may be injected and concentrated under conditions which enrich        and concentrate RNA for gene expression studies. The        concentrated RNA could be transcribed into cDNA by        reverse-transcription PCR and analyzed by microarray analysis or        SAGE, for example.

While a number of exemplary aspects and embodiments have been discussedabove, those of skill in the art will recognize that certainmodifications, permutations, additions and sub-combinations thereof areuseful. It is intended that the following appended claims and claimshereafter introduced are interpreted to include all such modifications,permutations, additions and sub-combinations as are within their truespirit and scope.

1. A method for concentrating selected particles, the method comprising:providing particles, including the selected particles, in a firstregion; applying a first field directed to move at least the selectedparticles from the first region into a second region, at least thesecond region being a region of a medium in which a mobility of theselected particles is dependent on a magnitude of one or more secondfields; and, when the selected particles are in the second region,concentrating the selected particles in a vicinity of a point in thesecond region by applying the second fields.
 2. A method according toclaim 1 comprising applying the first and second fields simultaneously.3. A method according to claim 1 comprising applying the first andsecond fields in alternation.
 4. A method according to claim 1 whereinapplying the one or more second fields overlaps in time with applyingthe first field.
 5. A method according to claim 1 comprisingreplenishing the first region with additional particles, includingadditional selected particles, applying the first field to move at leastthe additional selected particles from the first region into the secondregion; and, when the additional selected particles are in the secondregion, concentrating the additional selected particles together withthe selected particles by applying the one or more second fields.
 6. Amethod according to claim 1 wherein the first region comprises a regionof a liquid and the method comprises introducing a liquid samplecontaining the particles into the first region.
 7. A method according toclaim 6 wherein the medium comprises a gel and the method comprisesdriving the selected particles from the liquid into the gel by applyingthe first field.
 8. A method according to claim 1 wherein the selectedparticles are electrically charged and the first field comprises anelectric field.
 9. A method according to claim 1 wherein applying thefirst field results in moving some non-selected particles into thesecond region and the method comprises: moving the non-selectedparticles through and out of the second region by applying the firstfield; and, keeping the selected particles within the second region byintermittent or continuous application of the one or more second fields.10. A method according to claim 9 comprising, after moving at least theselected particles from the first region into the second region,removing particles from the first region and continuing to apply thefirst field for a period sufficient to substantially remove thenon-selected particles from the second region.
 11. A method according toclaim 10 wherein removing particles from the first region comprisesflushing the first region with a liquid that does not containnon-selected particles.
 12. A method according to claim 11 wherein theliquid comprises an aqueous buffer solution.
 13. A method according toclaim 9 comprising applying the one or more second fields for acumulative time exceeding a characteristic time by at least a factor of2 wherein the characteristic time is a time required for concentratingselected particles in the second region by application of the one ormore second fields and the method comprises, after commencingconcentrating the selected particles and before completing concentratingthe selected particles, introducing more selected particles into thesecond region from the first region by application of the first field.14. A method according to claim 1 wherein applying the one or moresecond fields comprises applying a time-varying electric field in thesecond region.
 15. A method according to claim 14 wherein applying thetime-varying electric field comprises providing a first electrode on oneside of the first region and a second electrode on one side of thesecond region such that the first and second regions both lie betweenthe first and second electrodes, monitoring an electrical potential at alocation symmetrical about the second region relative to the secondelectrode and, controlling an electrical potential at the firstelectrode in response to the monitored electrical potential to cause themonitored electrical potential to have a desired value.
 16. A methodaccording to claim 14 comprising, prior to applying the one or moresecond fields, applying the first field for a period, T_(INJ), thatsatisfies: $T_{INJ} \geq \frac{t_{SCODA}E_{SCODA}}{E_{DC}}$ where:t_(SCODA) is a length of time in one cycle of the one or more secondfields in which the one or more second fields have a direction oppositeto a direction of the first field; E_(SCODA) is a strength of the one ormore second fields when they have a direction opposite to a direction ofthe first field; and E_(DC) is a strength of the first field.
 17. Amethod according to claim 1 wherein the selected particles compriseparticles of a plurality of species and the method comprises, afterconcentrating the selected particles in the vicinity of the point,separating different species of the selected particles from one another.18. A method according to claim 17 wherein separating different speciesof the selected particles from one another comprises applying anasymmetrical time-varying electrical field to the selected particles andthereby causing the different species of selected particles to move atdifferent velocities in a direction determined by the time-varyingelectrical field.
 19. A method according to claim 18 wherein theasymmetrical time-varying electrical field comprises a sum of aunidirectional bias field component and an alternating field componenthaving a magnitude that integrates to zero over an integral number ofcycles of the alternating field component.
 20. A method according toclaim 17 comprising, prior to separating the different species of theselected particles, providing a spot comprising a mixture of specieshaving known characteristics on the medium and separating the specieshaving known characteristics simultaneously with separating thedifferent species of the selected particles.
 21. A method according toclaim 17 wherein separating the different species of selected particlescomprises causing a desired one of the plurality of species to remain inthe vicinity of the point while causing other ones of the differentspecies to move away from the vicinity of the point.
 22. A methodaccording to claim 17 comprising, prior to separating the differentspecies of the selected particles, providing a spot comprising a specieshaving known characteristics on the medium, wherein separating thespecies comprises applying one or more separation fields to the medium;monitoring a position of the spot; and controlling the one or moreseparation fields in response to the position of the spot.
 23. A methodaccording to claim 22 comprising controlling the one or more separationfields so as to maintain the spot stationary.
 24. A method according toclaim 1 wherein providing particles, including the selected particles,in the first region comprises collecting a sample of liquid and placingthe sample of liquid in the first region.
 25. A method according toclaim 1 wherein providing particles, including the selected particles,in the first region comprises rinsing an object to which the selectedparticles are attached in a liquid and then placing the liquid in thefirst region.
 26. A method according to claim 25 wherein the objectcomprises an air filter.
 27. A method according to claim 25 wherein theobject comprises a tissue sample.
 28. A method according to claim 25wherein the selected particles are entrained in a biological material onthe object and the method comprises washing some of the biologicalmaterial off of the object with the liquid.
 29. A method according toclaim 25 wherein the liquid comprises an aqueous buffer solution.
 30. Amethod according to claim 1 wherein the selected particles comprisemolecules of DNA.
 31. A method according to claim 30 comprising, afterconcentrating the selected particles, extracting the selected particlesfrom the medium.
 32. A method according to claim 30 comprisingamplifying the selected particles by means of the polymerase chainreaction.
 33. A method according to claim 1 wherein the selectedparticles comprise molecules of RNA.
 34. A method according to claim 1wherein the selected particles comprise protein molecules.
 35. A methodaccording to claim 34 comprising denaturing the protein molecules priorto applying the first field.
 36. A method according to claim 1 whereinthe selected particles comprise electrically-charged molecules.
 37. Amethod according to claim 36 wherein the charged molecules comprisepolynucleotides or oligonucleotides.
 38. A method according to claim 1wherein the first region comprises a region of a gel.
 39. A methodaccording to claim 1 wherein providing the particles in the first regioncomprises preparing a cell lysate and introducing the cell lysate intothe first region.
 40. A method according to claim 1 wherein providingthe particles in the first region comprises mixing a sample of soil witha liquid and introducing the liquid into the first region.
 41. A methodaccording to claim 1 wherein the first region comprises a chamberconnected to receive samples of a fluid containing the particles and themethod comprises changing the fluid in the chamber.
 42. A methodaccording to claim 41 comprising causing the fluid containing theparticles to flow through the chamber.
 43. A method for concentratingparticles of interest, the method comprising: driving the particles intoa medium by applying a particle-injecting electric field across aboundary between the medium and a sample containing the particles ofinterest; and, applying scodaphoresis to the particles of interest inthe medium to concentrate the particles of interest at a location in themedium.
 44. A method according to claim 43 wherein theparticle-injecting electric field is a DC field.
 45. A method accordingto claim 43 or 44 wherein applying the particle-injecting DC field andapplying scodaphoresis are performed concurrently.
 46. A methodaccording to claim 43 wherein applying the particle-injecting DC fieldand applying scodaphoresis are performed in alternation.
 47. A methodaccording to claim 43 wherein applying scodaphoresis comprises applyingat least one scodaphoresis electric field to the medium.
 48. A methodaccording to claim 47 wherein applying the particle-injecting field andapplying the scodaphoresis electric field are performed using the sameelectrodes.
 49. A method according to claim 43 wherein the mediumcomprises a gel.
 50. A method according to claim 49 wherein the mediumcomprises an agarose gel.
 51. A method according to claim 43 wherein theparticles of interest comprise electrically-charged molecules.
 52. Amethod according to claim 51 wherein the charged molecules comprisepolynucleotides or oligonucleotides.
 53. Apparatus for concentratingparticles of interest, the apparatus comprising: a buffer reservoircapable of receiving a sample; a medium in which particles of interesthave a mobility that depends upon the magnitude of applied fields; meansfor applying a first field to drive particles of interest from thebuffer reservoir into the medium; and, means for applying one or moresecond fields to concentrate the particles of interest at a focal spotwithin the medium.
 54. Apparatus according to claim 53 comprising a pumpconnected to refresh a sample in the buffer reservoir.
 55. Apparatusaccording to claim 54 comprising an electrode in the buffer reservoirand a sensing electrode in the buffer reservoir between the electrodeand the medium.
 56. Apparatus according to claim 55 wherein the sensingelectrode is connected to a controller and the controller is configuredto control a potential applied to the electrode to cause a potential atthe sensing electrode to vary in time according to a desired SCODAwaveform. 57-58. (canceled)